The subject invention relates to a nucleic acid based diagnostic system for rapid and specific flavivirus virus identification and quantification and kits which involve a novel set of oligonucleotides which can distinguish and quantitate flavivirus, especially.
Dengue viruses belong to the family Flaviviridae, which contains almost 70 viruses, including those causing yellow fever and several encephalitides. Four distinct dengue virus types (dengue 2, 3, and 4) are each capable of causing infection in humans (Henchal and Putnak. 1990). Dengue infections are usually confirmed by serologic detection of dengue-specific antibodies or/and dengue virus isolation through culture in insect cells or mosquito inoculation (Gubler, 1989). These conventional methods for dengue virus detection are very time consuming, labor intensive, and have limited sensitivity for detecting low levels of dengue virus. Development of sensitive, type-specific dengue diagnostic systems is crucial because of explosive growth in dengue virus transmission and the increasing incidence of dengue hemorrhagic fever (Monath, 1994).
Rapid molecular diagnostic systems, such as RT-PCR, could provide a possible solution for dengue virus identification from clinical samples (Harris et al., 1998). Several RT-PCR systems for dengue virus detection have been reported, based on different conserved genomic regions, such as genes for nonstructural proteins (Chow et al., 1993; Fulop et al., 1993) and the 3xe2x80x2-noncoding region (Sudiro et al., 1997). See also U.S. Pat. No. 5,939,254. However, these methods frequently have mismatches corresponding to their amplicons, or even to primer sequences, due to genetic differences among strains of the same dengue serotype. Some of these systems rely upon sequences from the xe2x80x9cconservedxe2x80x9d regions for probes or primers, such as NS3 and NS5 genes. Alignment of these so-called conserved genes of dengue reveal mismatches among the viruses, in addition to variation among virus strains. These mismatches result in reduced sensitivity of the assay, and as a result, false diagnosis of the specific dengue virus. Further, unpredictable variations in PCR target sequences would yield lower specificity and sensitivity in dengue detection. In addition, these methods are often cumbersome to adapt for routine clinical use and are not quantitative.
The 3xe2x80x2 noncoding region has been previously studied and sequenced. See, for example, Mandl, C. W., et al, xe2x80x9cSpontaneous Engineered Deletions in the 3xe2x80x2 noncoding region of Tick-borne Encephalitas Virus: Construction of Highly Atenuated Mutants of a Flavivirusxe2x80x9d, J. Virology, Vol. 72, (1998) pp. 2132-2140. Proupski, V. et al., xe2x80x9cBiological Consequences of Deletions within the 3xe2x80x2-untranslated Region of Flavivirus May be due to Rearrangements of RNA Secondary Structurexe2x80x9d, Virus Research (1999) Vol. 64, pp. 107-123.
The invention herein disclosed addresses these and other associated problems.
The present invention is directed to designing and utilizing specific genomic region, the conserved 3xe2x80x2-terminal non-coding region, as specific nucleic acid-based diagnostic system for rapid and specific flavivirus, e.g. dengue virus, identification and quantification. In this regard, the invention focuses on the last 250, more specifically the last 200, even more specifically the last 160 bases of the conserved dengue genome corresponding to nucleotides 10558 through 10718 of dengue 1. The invention also utilizes flavivirus specific upstream primers, probes, and downstream primers developed from this region.
From the conserved terminal 3xe2x80x2 non coding region conserved region, a probe unique to a flavivirus is chosen. Primers can then be generated from conserved areas upstream and downstream of the probe that are unique to the virus of interest, the areas extending to about one kilobase from the 3xe2x80x2 terminus of any flavivirus. More than one primer set and more than one probe can be chosen, each primer set and probe can be both conserved and specific for a virus.
Once the cDNA is isolated from the samples containing the viruses to be identified, the cDNA is incubated with the primers and probe under condition such that a polymerase is able to synthesize a complementary strand using the primer/template substrate. The polymerase, having a 5xe2x80x2-to 3xe2x80x2 exonuclease activity, will digest the probe hybridized to its specific complementary virus sequence. The probe may contain a reporter at the 5xe2x80x2 end which will release a detectable signal when the probe is digested. Quantification and detection of the specific virus for which the probe and primers were designed is possible by detecting the released probe signal. If the probe does not find its match in the sample, the probe is not digested by the polymerase, and the signal is not released or detected.
The advantages of this invention are that probes can be highly discriminatory, even single nucleotide substitutions can be reliably detected among different viruses.
Oligonucleotide probes and primers described in this invention can be used in a multiplex format to detect and differentiate flaviviruses. It is also possible to formulate individual virus-specific assay mixtures to identify and quantitate one virus at a time. The format of the assay depends of the desired usage. A generic dengue diagnostic system may work well for clinical or epidemiologic use, whereas stereotype-specific dengue assays are required for use in vaccine development. The design of the assay can be refined for single-step multiplex use (processing of a specimen and evaluation in a single reaction mixture), or two step reactions may be used, as described in the material.
The flavivirus fluorogenic assays based on the terminal 3xe2x80x2 noncoding region offer real-time quantitation of virions (expressed as plaque forming units or as genome copy equivalents). Determination of viral number is extrapolated from reproducible linear standard curves, derived either from titration of viral RNA or from dilutions of flaviviral cDNA (as plasmids). The rapid, sensitive detection of flaviviruses lends itself to several applications. These include:
1. Rapid diagnosis of flaviviral infection: the specific nature of the fluorogenic probes devised for this patent allows confirmation of flaviviral infection. Use of selected flaviviral probes (for example, for a particular geographic region) would support clinical/epidemiologic efforts to quickly and accurately establish the cause of specific illnesses such as febrile illness. Typically, the diagnostic apparatus required to confirm specific flaviviral infection is cumbersome and restricted to reference laboratories. The new methods allow rapid, bedside diagnosis and confirmation of flavivirus infection. This use will be greatly facilitated by the availability of portable field-tested diagnostic platforms during future deployments to endemic regions.
2. Determination of viral burden: the magnitude and duration of viremia during flaviviral infection may be positively associated with flaviviral pathogenecity. This finding may be of prognostic significance in dengue hemorrhagic fever, where increased viral burden (particularly at the time of defervescence) may be a marker of increased risk for severe disease. Studies of viral clearance by different immunologic mechanisms will be made easier. Furthermore, the effectiveness of specific antiviral therapy (such as drugs, immunotherapy, and other approaches) may be gauged by its effect on viral burden in vivo or in vitro.
3. Quality control of flaviviral biologics: the precise and specific determination of viral titer in flaviviral vaccines and other biologics is a difficult and time-consuming effort, often requiring three separate bioassays. The availability of a nucleic acid-based assay that is both specific and sensitive provides a significant advance in analysis (identification and characterization) of biologics, central to the quality assurance components of the manufacturing documentation required for any vaccine. Moreover, this methodology may be used with both live, recombinant, and killed flaviviral vaccines; it is also applicable to complex combinations of flaviruses, such as multivalent dengue vaccines.
4. Amplification of flaviviral RNA: the described method offers a simplified approach to creation of amplified, near full-length copies of flaviviral genomes. The 3xe2x80x2 noncoding region is critical to replication of viral RNA, and the product of the reverse transcription reaction could be manipulated to generate flaviviral replicons. In addition, formation of replicative intermediates can be monitored through antisense RNA copies. Flaviviral cDNA products may be used for molecular pharmacologic or therapeutic purposes.